Rotaviruses are the major etiological agents of severe infantile diarrhea and nearly all humans are infected by these viruses within the first 3 years of life. The mortality due to rotavirus infection is significant, globally causing nearly 1 million deaths each year. The development of vaccines against these viruses is complicated by the existence of four major rotavirus serotypes that cause disease and co-circulate throughout the world. Other serotypes have been identified which on a more regional basis are also important causes of diarrhea. Recently, a tetravalent vaccine was developed in the Laboratory of Infectious Diseases which provides a high level of protection against severe rotaviral diarrhea. Because of the pathogenic importance, an overall goal of the Laboratory remains the development of new vaccines, the improvement of existing vaccines, and the identification of other methods for preventing and treating rotaviral disease. Accomplishing this goal would be helped by a more complete understanding of the molecular biology of the rotavirus, which is the basis of this project. In particular by defining events in the replication and packaging of the rotavirus genome, we should be able to identify targets in the viral genome that when mutagenized will lead to the attenuation of virulent human rotaviruses and the generation of vaccine candidates. Despite extensive efforts, it has not yet been possible to develop a reverse genetics system that allows us to introduce such mutations into the rotavirus genome. The goal of this project is to characterize the fundamental events in the replication of the segmented double-stranded (ds)RNA genome of rotaviruses and then to apply this knowledge to the establishment of a reverse genetics system and the development of rotavirus vaccines. The rotavirus core consists of three proteins: the RNA polymerase VP1, the capping enzyme VP3, and the core shell proteins VP2, and eleven segments of dsRNA. Virion-derived cores have replicase activity in vitro, and this activity has been used to probe the RNA replication process. In the infected cell, the packaging and replication of the eleven viral mRNAs are coordinately replicated and packaged. This process requires each viral mRNA to have at least two sets of recognition signals: one set of common signals used by the viral RNA polymerase to recognize all eleven viral mRNAs and another set that is unique for each of the mRNAs and must function in packaging and assortment. This project will describe the cis-acting signals in the eleven viral mRNAs that particpate in packaging, assortment and minus strand synthesis and will delineate the specificity and function of the viral RNA-binding. An ultimate goal of this project is to incorporate its findings into the development of a cell-free system which allows the assembly of infectious particles from recombinant protein and RNA. Multiple experimental approaches will be used to accomplish the aims of this project. (i) The replicase activity of open cores will be used in combination with cDNA-derived mutant RNAs to identify cis-acting signals in viral mRNAs that are important for genome replication, (ii) Computer modeling and biochemical mapping will be used to characterize the structure of the cis- acting replication signals of the mRNAs, (iii) Recombinant proteins will be used to assess the function of individual proteins in RNA recognition, packaging, assortment and replication, and (iv) RNA-binding assays (gel mobility shift assays) will be used to examine the specificity and targets of the rotavirus RNA-binding proteins.